Formulations useful against hepatitis C virus infections

ABSTRACT

The present invention relates generally to chemical compounds and substances which are effective against Hepatitis C virus (HCV) infections. Moreover, the present invention relates to compositions comprising said compounds and/or substances, to methods for preventing HCV infections as well use of the compounds and/or substances for the preparation of compositions useful for the prophylaxis and/or treatment of HCV infections. Useful compounds and substances according to the invention are selenium, selenium salts, Vitamin D 3  and retinoids, like all trans retinoic acid and salts thereof, C 1 -C alkyl amides of all trans retinoic acid and salts thereof, C 1 -C 10  alkyl esters of all trans retinoic acid and salts thereof, 9-cis retinoic acid and salts thereof, C 1 -C 10  alkyl amides of 9-cis retinoic acid and salts thereof, C 1 -C 10  alkyl esters of 9-cis retinoic acid and salts thereof, (E)-4-[2(5,6,7,8-tetrahydro-5,5,8,8-tetra methyl-2-naphthalenyl-1-propenyl)benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN).

The present invention relates to chemical compounds and substances whichare effective against Hepatitis C virus (HCV) infections. In particular,the present invention relates to compositions comprising said compoundsand/or substances, to methods for preventing HCV infections as well asuse of the compounds and/or substances for the preparation ofcompositions useful for the prophylaxis and/or treatment of HCVinfections.

BACKGROUND OF THE INVENTION

Hepatitis C Virus (HCV) infection Is a major cause of chronic hepatitis,cirrhosis and hepatocellular carcinoma. The WHO estimates thatapproximately 3% of the world population, or 170 million people, havebeen infected with the Hepatitis C Virus. In the U.S., an estimated 3.9million Americans have been infected (CDC fact sheet September 2000).Over 80% of HCV-infected individuals develop chronic hepatitis, which isassociated with disease states ranging from asymptomatic carrier statesto repeated inflammation of the liver and serious chronic liver disease.Over the course of 20 years, more than 20% of chronic HCV-patients areexpected to be at risk to develop cirrhosis or progress tohepatocellular carcinoma. Liver failure from chronic hepatitis C is theleading indicator for liver transplantation. Excluding transplantation,the CDC estimates that medical and work-loss cost for HCV annually arearound US-$ 600 million. HCV is transmitted primarily by blood and bloodproducts. Due to routine screening of the blood supplies from mid-1992,new transfusion-related cases are exceedingly rare and have beensurpassed by injection drug use as the highest risk factor for acquiringthe virus. There is also a sexual, however inefficient, route oftransmission, and a 6% rate of transmission from infected mothers totheir children, which is higher in case of HIV co-infection. In acertain percentage of infections, the mode of transmission remainsunknown. In spite of the significant decline in incidence in the 1990's,the number of deaths (estimated deaths annually at the moment: 8000 to10,000 in U.S.) and severe disease due to HCV is anticipated to triplein the next 10 to 20 years (sources: CDC fact sheet (accessed Dec. 12,2000); Houghton M.

Hepatitis C Viruses. In B N Fields, D M Knipe, P M Howley (ed.) FieldsVirology. 1996. Lippencott-Raven Pub., Philadelphia; Rosen H R andGretch D R, Molecular Medicine Today Vol 5, 393, September 99; Science285, 26, July 99: News Focus: The scientific challenge of Hepatitis C;Wong J B et al, Am J Public Health, 90, 1562, October 2000: Estimatingfuture. hepatitis C morbidity, mortality, and costs in the UnitedStates).

According to the announcement from the EASL (European Association forthe Study of the Liver) International Consensus Conference on HepatitisC (Feb. 26-28, 1999, Paris, France), combination therapy of alphainterferon and ribavirin is the recommended treatment for naivepatients. Monotherapy with interferon has also been approved by the FDA,but the sustained response rate (HCV RNA remains undetectable in theserum for more than 6 months after end of therapy) is only 15 to 20%, incontrast to 35 to 45% with combination therapy. Interferons (Intron A,Schering-Plough; Roferon A, Hoffmann-LaRoche; Wellferon, Glaxo Wellcome;Infergen, Amgen) are injected subcutaneously three times a week,ribavirin (Rebetol, Schering-Plough) is an oral drug given twice a day.Recommended treatment duration is 6 to 12 months, depending on HCVgenotype. Experimental forms of slow-release pegylated interferons(Pegasys, Hoffmann-LaRoche; PEG-Intron, Schering-Plough) have shownimprovements in response rates (42 to 82% in combination with ribavirin)and application (once-weekly injection) in recent clinical studies(Hepatology 32:4, Pt 2 of 2. October 2000; NEJM 343, 1673. December2000; NEJM 343, 1666. December 2000). Common side effects of interferontherapy include: e.g. fatigue, muscle aches, head aches, nausea, fever,weight loss, irritability, depression, bone marrow suppression,reversible hair loss. The most common side effects of ribavirin areanemia, fatigue and irritability, itching, skin rash, nasal stuffiness,sinusitis, cough. More serious side effects of mono- and combinationtherapy occur in less than two percent of patients (NIDDK information:Chronic Hepatitis C: Current Disease Management. accessed Sep. 12,1999). Some of the contraindications to interferon are psychosis orsevere depression; neutropenia and/or thrombocytopenia; organtransplantation except liver; symptomatic heart disease; decompensatedcirrhosis; uncontrolled seizures. Contraindications to ribavirin areend-stage renal failure; anemia; hemoglobinopathies; severe heartdisease; pregnancy; no reliable method of contraception (consensusstatement EASL). Moreover, treatment of Hepatitis C virus infection withinterferon-alpha is effective in only a minority of individuals. Thissuggests that the virus may use various tricks to be resistant tointerferon.

Review articles by Vogel, “Peginterferon-α_(2a) (40 kDa)/ribavirincombination for the treatment of chronic hepatitis C infection”, ExpertRev. Anti-infect. Ther. 1 (3), 423-431 (2003) and Durantel et al.,“Current and emerging therapeutic approaches to hepatitis C infection”,Expert Rev. Anti-infect. Ther. 1 (3), 441-454 (2003) provide an overviewof the current status of HCV therapy. According to these articles,current treatment involves association of two molecules, standard (i.e.non-pegylated) or pegylated interferon and ribavirin. Although thistherapy induces a sustained virologic response in 50 to 60% of thecases, there are still a high number of so-called ‘non-responders’ andtreatment is often limited by the above-mentioned side effects.Particularly for non responding patients, and more generally, forimproving the current clinical practice, it is important to developalternative and effective therapeutic approaches for HCV treatment.

If in the following the terms “non-responder(s) to interferon and/orribavirin therapy” or “non responding patient(s) to interferon and/orribavirin therapy” are used, these terms shall denote the portion of theHCV infected individuals (particularly humans) who do not show apositive reaction or total cure when treated with pegylated ornon-pegylated (standard) α-, β-, or γ-interferon alone (so-calledinterferon monotherapy), ribavirin alone (so-called ribavirinmonotherapy), or a combination therapy of pegylated or non-pegylated(standard) α-, β-, or γ-interferon and ribavirin. Non-responders can bepatients who are non responding to interferon and/or ribavirin treatmentfrom the very beginning of a therapy, or who become non responding aftera certain time of an interferon and/or ribavirin treatment.

Experimental treatments that are not new forms of Interferon areMaxamine (histamine dihydrochloride, Maxim Pharmaceuticals), which willbe combined with Interferon in phase III studies, VX-497 (VertexPharmaceuticals), an IMP dehydrogenase inhibitor, as a less toxicribavirin substitute in phase II, and amantadine (Endo Labs), anapproved influenza drug, as the third component in triple therapy (phaseII). Inhibitors for HCV enzymes such as protease inhibitors, RNAdependent RNA polymerase inhibitors, helicase inhibitors as well asribozymes and antisense RNAs are under preclinical development(Boehringer Ingelheim, Ribozyme Pharmaceuticals, Vertex Pharmaceuticals,Schering-Plough, F Hoffmann-LaRoche, Immusol, Merck etc.). No vaccine isavailable for prevention or therapeutic use, but several companies aretrying to develop conventional or DNA vaccines or immunostimulatoryagents (e.g. Chiron, Merck/Vical, Epimmune, NABI, Innogenetics). Inaddition, antibodies against HCV virion have been developed and enteredinto clinical trials recently (Trimera Co., Israel).

In summary, the available treatment for chronic Hepatitis C isexpensive, effective only in a certain percentage of patients andadverse side effects are not uncommon.

WO 02/066022 A1 describes a method of treating hepatitis comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of retinoid such as all-trans retinoic acid. Inparticular embodiments, the form of hepatitis is Hepatitis A, B, C, D, Eand G and the treatment is with liposomal all-trans retinoic acid.

DESCRIPTION OF THE INVENTION

Recent research has revealed how cells communicate with each other tocoordinate the growth and maintenance of the multitude of tissues withinthe human body. A key element of this communication network is thetransmission of a signal from the exterior of a cell to its nucleus,which results in the activation or suppression of specific genes. Thisprocess is called signal transduction.

Signal transduction at the cellular level refers to the movement ofsignals from outside the cell to inside. The movement of signals can besimple, like that associated with receptor molecules of theacetylcholine class: receptors that constitute channels which, uponligand interaction, allow signals to be passed in the form of small ionmovement, either into or out of the cell. These ion movements result inchanges in the electrical potential of the cells that, in turn,propagates the signal along the cell. More complex signal transductioninvolves the coupling of ligand-receptor interactions to manyintracellular events. These events include phosphorylations by tyrosinekinases and/or serine/threonine kinases. Protein phosphorylations changeenzyme activities and protein conformations. The eventual outcome is analteration in cellular activity and changes in the program of genesexpressed within the responding cells.

Signal transducting receptors are of three general classes:

1. Receptors that Penetrate the Plasma Membrane and have IntrinsicEnzymatic Activity:

Receptors that have intrinsic enzymatic activities include those thatare tyrosine kinases (e.g. PDGF, insulin, EGF and FGF receptors),tyrosine phosphatases (e.g. CD45 [cluster determinant-45] protein of Tcells and macrophages), guanylate cyclases (e.g. natriuretic peptidereceptors) and serine/threonine kinases (e.g. activin and TGF-betareceptors). Receptors with intrinsic tyrosine kinase activity arecapable of autophosphorylation as well as phosphorylation of othersubstrates.

Additionally, several families of receptors lack intrinsic enzymeactivity, yet are coupled to intracellular tyrosine kinases by directprotein-protein interactions. This class of receptors includes all ofthe cytokine receptors (e.g. the interleukin-2 receptor) as well as theCD4 and CD8 cell surface glycoproteins of T cells and the T cell antigenreceptor.

2. Receptors that are Coupled, Inside the Cell, to GTP-Binding andHydrolyzing Proteins (Termed G-Proteins):

Receptors of the class that interact with G-proteins all have astructure that is characterized by seven transmembrane spanning domains.These receptors are termed serpentine receptors. Examples of this classare the adrenergic receptors, odorant receptors, and certain hormonereceptors (e.g. glucagon, angiotensin, vasopressin and bradykinin).

3. Receptors that are Found Intracellularly and Upon Ligand BindingMigrate to the Nucleus where the Ligand-Receptor Complex DirectlyAffects Gene Transcription:

The steroid/thyroid hormone receptor superfamily (e.g. glucocorticoid,vitamin D, retinoic acid and thyroid hormone receptors) is a class ofproteins that reside in the cytoplasm and bind the lipophilicsteroid/thyroid hormones. These hormones are capable of freelypenetrating the hydrophobic plasma membrane. Upon binding ligand thehormone-receptor complex translocates to the nucleus and bind tospecific DNA sequences resulting in altered transcription rates of theassociated gene.

When the message reaches the nucleus via one or several of the pathwaysdescribed above, it initiates the modulation of specific genes,resulting in the production of RNA and finally proteins that carry out aspecific biological function. Disturbed activity of signal transductionmolecules may lead to the malfunctioning of cells and disease processes.Specifically, interaction of HCV with host cells is necessary for thevirus to replicate.

The present invention is based upon the fact that the human cellularprotein glutathione peroxidase-gastrointestinal (P18283) is specificallydownregulated as a result of HCV replication in HCV infected host cells.The antiviral prophylactic and/or therapeutic approach described hereinfocuses on specific chemical substances and compounds that can be usedto upregulate the human cellular protein glutathioneperoxidase-gastrointestinal. These specific chemical substances andcompounds are selenium, selenium salts, Vitamin D₃, pegylated andnon-pegylated (standard) α-, β-, and γ-interferon, ribavirin, andretinoids, particularly all isomeric forms of retinoic acid, like alltrans retinoic acid, salts of all trans retinoic acid, C₁-C₁₀ alkylesters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of alltrans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid,salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoicacid, salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cisretinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid,C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amidesof 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoicacid, C₁-C₁₀ alkyl esters of 13-cis retinoic acid, salts of C₁-C₁₀ alkylesters of 13-cis retinoic acid, C₁-C₁₀ alkyl amides of 13-cis retinoicacid, salts of C₁-C₁₀ alkyl amides of 13-cis retinoic acid, as well as(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoicacid (TTNPB),(4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)carboxamido]benzoicacid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR) and6-[3-1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437;AHPN).

According to a further aspect of the present invention, it is preferredthat together with one or more of the above-mentioned substancesparaquat is used as antiviral prophylactic and/or therapeutic substancethat can be used to upregulate the human cellular protein glutathioneperoxidase-gastrointestinal.

Preferred is the use of all trans retinoic acid or 13-cis retinoic acidfor the treatment of non-responders. Also preferred is the use ofcompositions comprising all trans retinoic acid or 13-cis retinoic acidwith one of the chemical substances mentioned above for the treatment ofHCV infections or HCV infection related diseases. Particularly, (i) alltrans retinoic acid is used with selenium and/or selenium salts, (ii)all trans retinoic acid is used with pegylated and/or non-pegylated(standard) α-, β-, and γ-interferon, (iii) all trans retinoic acid isused with pegylated and/or non-pegylated (standard) α-, β-, and/orγ-interferon and ribavirin, (iv) all trans retinoic acid is used withpegylated and/or non-pegylated (standard) α-, β-, and/or γ-interferonand with selenium and/or a selenium salt, (v) all trans retinoic acid isused in combination with ribavirin and selenium and/or a selenium salt,and (vi) all trans retinoic acid and ribavirin. In case of combination(v), all trans retinoic acid is preferably used at a concentration of0.1 to 10 μM, more preferably 0.5 to 2.5 μM, and particularly 1 μM,selenium or selenium salts are preferably used at a concentration of 1to 200 nM, more preferably 10 to 100 nM, and particularly 50 nM,ribavirin is preferably used at a concentration of 1 to 500 μM, morepreferably 10 to 100 μM, and particularly 50 μM. The above-mentionedcombinations (i), (ii), (iii), (iv), and (v) can be used both for thetreatment of responders and non-responders.

In addition, the following compounds shall be encompassed by the term“retinoids” as used within the present application. In particular, thefurther retinoids as understood according to the present applicationalso refer to retinol, etretinate, amides of the all-trans-retinoic acidor 13-cis-retinoic acid with 2-aminoethanol, alpha-L-serine,alpha-L-threonine, alpha-L-tyrosine containing phosphate groups.

The structure of these further retinoids is covered by the followinggeneral formula:R—CONH—X—OPO(OH)₂,

wherein

R is

and X is—CH₂—CH₂or—CH(CO₂H)—CH₂—or—CH(CO₂H)—CH(CH₃)—or—CH(CO₂H)—CH₂—C₆H₅—

Thus the amino group of 2-aminoethanol or the alpha-amino group of aminoacid forms an amide bond with the carboxylic group of all-trans-retinoicacid or 13-cis retinoic acid. At the same time the hydroxyl group of2-aminoethanol and the amino acid is modified by a phosphate residue.

Retinoids falling under the further retinoids according to the presentInvention are e.g. described In U.S. Pat. No. 6,326,397 B1 and U.S. Pat.No. 6,403,554 B2, which are incorporated herein by reference in theirentirety. In these two US patents, among other substances amides ofall-trans-retinoic acid or 13-cis-retinoic acid with 2-aminoethanol,alpha-L-serine, alpha-L-threonine, alpha-L-tyrosine are disclosed. Atthe same time hydroxyl groups of amino acids and 2-aminoethanol aremodified by phosphate residues. The all-trans-retinoic acid or 13-cisretinoic acid have been derived by various procedures fromnaturally-occurring products. It is however possible, within the scopeof the present invention, to produce these compound synthetically.Examples how to synthesize the further retinoids according to thepresent invention are described in the above-mentioned patents U.S. Pat.No. 6,326,397 B1 and U.S. Pat. No. 6,403,554 B2.

The main characteristic among the synthesised compounds is thephosphorylation of the hydroxyl groups of N-acyl derivatives of aminoacids and 2-aminoethanol.

Further retinoids (retinoic acid derivatives) according to the presentinvention include the following compounds:

1. N-(all-trans-retinoyl)-o-phospho-2-aminoethanol

1a. N-(13-cis-retinoyl)-phospho-2-aminoethanol

2. N-(all-trans-retinoyl)-phospho-L-serine

2a. N-(13-cis-retinoyl)-phospho-L-serine

3. N-(all-trans-retinoyl)-o-phospho-L-threonine

3a. N-(13-cis-retinol)-o-phospho-L-threonine

4. N-(all-trans-retinoyl)-o-phospho-L-tyrosine

4a. N-(13-cis-retinoyl)-o-phospho-L-tyrosine

The structural formulas of the above-mentioned compounds are presentedbelow:

Moreover, also the retinoid antagonists described in U.S. Pat. No.6,326,397 B1 do fall under the retinoids according to the presentinvention. The contents of U.S. Pat. No. 6,326,397 B1 is herewithincorporated in its entirety into the present application. Specifically,the present application also relates to compounds of the formula I

wherein the dotted bond can be either hydrogenated or form a doublebond; and, when the dotted bond forms a double bond, R¹ is lower alkyland R² is hydrogen; and, when the dotted bond is hydrogenated, R¹ and R²taken together are methylene to form a cis-substituted cyclopropyl ring;R³ is hydroxy or lower alkoxy; R⁴ is alkyl or alkoxy; and R⁵ and R⁶ are,independently, a C₄-C₁₂ alkyl or a 5-12 cycloalkyl substituentcontaining from 1-3 rings which are either unsubstituted or substitutedwith from 1-3 lower alkyl groups, with the carbon atom of R⁵ and R⁶being linked to the remainder of the molecule to form a quaternarycarbon atom and pharmaceutically acceptable salts of carboxylic acids offormula I.

As used herein the term “alkyl” means straight-chain, branched or cyclicalkyl residues, in particular those containing from 1 to 12 carbonatoms, such as methyl, ethyl, propyl, isopropyl, t-butyl, decyl,dodecyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. The term“lower alkyl” means alkyl groups containing from 1 to 7, preferably 14carbon atoms. Most preferred lower alkyl groups are methyl and ethyl.Alkyl and alkoxy groups denoted by R⁴ preferably contain 18 carbonatoms, more preferably 14 carbon atoms. Particularly preferred group R⁴are ethoxy and butoxy. Examples of C₄₋₁₂ alkyl groups represented by R⁵or R⁶ are tert.-butyl, 1,1-dimethylpropyl, 1-methyl-1-ethylpropyl,1-methyl-1-ethylhexyl and 1,1-dimethyldecyl. Of these groups,tert.-butyl is preferred. When one of R⁵ and R⁶ is a 5 to 12 cycloalkylhydrocarbon substituted, the substituent contains from 1 to 3 fusedhydrocarbon rings which may be unsubstituted or substituted with from 1to 3 lower alkyl groups. The substituents R⁵ and R⁶ are attached to theremainder of the molecule of formula I by a carbon atom which, when soattached, forms a quaternary carbon atom. Among the preferred mono- orpolycyclic hydrocarbon substituents represented by R⁵ and R⁶ are1-adamantyl and. 1-methylcyclohexyl.

In one embodiment, the invention comprises compounds of the formula I a

wherein R¹ is lower alkyl and R³ to R⁶ are as in formula I; andpharmaceutically acceptable salts of carboxylic acids of formula Ia.

In another embodiment the invention comprises compounds of the formulaIb:

wherein R³ to R⁶ are as in formula I; and pharmaceutically acceptablesalts of carboxylic acids of formula Ib.

The compounds of formula I wherein R¹ and R² taken together aremethylene, may be present in pure enantiomeric form or as racemates.While formula Ib arbitrarily depicts a particular enantiomeric form itis to be understood that the invention also comprises the oppositeenantiomers as well as the racemates.

Particularly preferred are compounds of the formula Ia wherein R¹ ismethyl, R⁴ is ethoxy or butoxy and R⁵ and R⁶ are tert.-butyl.

The compounds of formula I above bind specifically to Retinoid XReceptors (RXR), but do not activate them. Accordingly the compounds ofthis invention can be used to reduce or abolish adverse events inducedby retinoids (retinoid agonists) in patients.

In a further aspect, the present Invention relates to the use ofretinoid antagonists comprising retinoids with selective Retinoic AcidReceptor (RAR) antagonistic activity, Retinoid X Receptor (RXR)antagonistic activity or mixed RAR-RXR antagonistic activity.

In accordance with that aspect of the invention the term “retinoidantagonists” is used for retinoids or compounds with RAR, RXR or mixedRAR-RXR antagonistic activity. It includes compounds with receptorneutral antagonistic activity (neutral antagonists), receptor inverseagonistic activity (inverse agonists) and negative hormone activity(negative hormones).

Thus, the term “retinoid antagonists” encompassesa) RXR antagonists of the formula I given earlier herein, particularlythose of the formula Ic

wherein the dotted bond is optional; and, when the doffed bond ispresent, R¹ is methyl and R² is hydrogen; and, when the dotted bond isabsent, R¹ and R² taken together are methylene to form a cis-substitutedcyclopropyl ring; and R⁴¹ is C₁₋₄-alkoxy;b) RAR α-antagonists of formulae

wherein R⁷ is C₅₋₁₀-alkyl, and R⁸ and R⁹ independently of each other arehydrogen or fluorine; such compounds being described in U.S. Pat. No.5,391,766 and J. Med. Chem. 1997, 40, 2445;c) RAR α,β antagonists of formulae

wherein R¹⁰ is diamantyl, X is O or NH, R¹¹ is phenyl or benzyl, andwherein optionally either ring A or ring B is present; such compoundsbeing described in Med. Chem. Res. 1991, 1, 220; Biochem. Biophys. Res.Com. 1997, 231, 243; J. Med. Chem. 1994, 37, 1508;d) RAR β,γ antagonists of formula

wherein R¹² and R¹³ independently of each other hydroxy, C₁₋₄-alkoxy,optionally branched C₁₋₅-alkyl or adamantyl; such compounds beingdescribed in J. Med. Chem. 1995, 38, 4993;e) RAR γ antagonists of formulae

such compounds being described in Cancer Res. 1995, 55, 4446;f) RAR α,β,γ antagonists of formulae

wherein Y is —CH₂— or sulfur and Z is —CH═ or nitrogen, and R¹⁴ ishydrogen or C₁₋₄-alkyl; such compounds being described in J. Med. Chem.1995, 38, 3163 and 4764; J. Biol. Chem. 1996, 271, 11897 and 22692;g) RXR antagonists of formula

wherein R¹⁵ is C₁₋₄-alkoxy; such compounds being described in J. Med.Chem. 1996, 39, 3229; and Nature 1996, 383, 450, as well aspharmaceutically acceptable salts and pharmaceutically acceptablehydrolyzable esters of the compounds of formulae III to XVII.

In the scope of the present invention, the “pharmaceutically acceptablesalts” includes any salt chemically permissible in the art for retinoidsand particularly retinoid antagonists and applicable to human patientsin a pharmaceutically acceptable preparation. Any such conventionalpharmaceutically acceptable salt of retinoids or retinoid antagonistscan be utilized. Among the conventional salts which can be utilized,there are the base salts included, for example, alkali metal salts suchas the sodium or potassium salt, alkaline earth metal salts such as thecalcium or magnesium salt, and ammonium or alkyl ammonium salts.

Moreover, the following substances disclosed in the commerciallyavailable database “Pharmaprojects” be considered to fall under the term“retinoids” according to the present invention. Originator Generic NameChemical Structure Allergan AGN-181701

Allergan AGN-192174

Allergan AGN-193676

Allergan AGN-193836

Allergan AGN-4326 Allergan AGN-194310

Allergan tazarotene

Allergan AGN-195183

Antigenics retinoic acid, Antigenics

AP Pharma trans-retinoic acid, AP Pharma

Barrier rambazole Therapeutics Basilea Pharmaceutica alitretinoin,Basilea

BattellePharma Isotretinoin, EHD delivery

Bristol-Myers Squibb BMS-297208

Bristol-Myers Squibb Pharmaprojects No. 6087

Bristol-Myers Squibb BMS-181163

Bristol-Myers PLT-99257 Squibb Clarion CPR-2003 Eisai ER-35794

Eisai polypreic acid

Eisai Pharmaprojects No. 5718

Eisai ER-34617

Galderma CD-437

Galderma adapalene

Hoffmann-La Roche etarotene

Hoffmann-La Roche etretinate

Hoffmann-La Roche issotertinoin

Hoffmann-La Roche motretinide

Hoffmann-La Roche Ro-11-0503

Hoffmann-La Roche Ro-13-6298

Hoffmann-La Roche Ro-23-2895

Hoffmann-La Roche sumarotene

Hoffmann-La R-667 Roche Hoffmann-La Roche Pharmaprojects No. 5126

Hoffmann-La Roche tertinoin, Roche

Hoffmann-La Roche Pharmaprojects No. 4858

Incyte MX6 Corporation Incyte Corporation MX-781

Johnson & Johnson Pharmaprojects No. 697

Johnson & Johnson Pharmaprojects No. 5849

Johnson & Johnson tretinoin, Ortho

Johnson & Johnson fenretinide

Ligand LGD-1550

Ligand alltretinoin, gel, Ligand

Ligand bexarotene, oral, Ligand

Ligand bexarotene, gel, Ligand

Ligand Pharmaprojects No. 4983

Ligand alitertinoin, oral, Ligand Ligand LY-929

Ligand LG-100754

Molecular Design MDI-101 Molecular Design MDI-301 Molecular DesignMDI-403 NIH trans-retinoic acid, NIH

NIH 9-cis-retinoic acid, NCI

Nisshin Pharma tocoretinate

Non-industrial source TTNN

Non-industrial source RBAD

Non-industrial source Pharmaprojects No. 2187

Oxford BioMedica RARβ2, gene ther, Oxford Bio Pilot TherapeuticsPLT-99511 Scotia Pharmaceuticals tertinoin, galactosome, Scotia

Shionogi AM-80

SRI International SRI-6409-40

SRI International Pharmaprojects No. 3749

SRI International Pgarmaprojects No. 472

SRI International Pharmaprojects No. 1168

Taiho TAC-101

UAB Research Foundation UAB-30

Yamanouchi clindamycin +teertinoin, Yaman

According to another embodiment of the present invention, furthersubstances can be combined with the at least one of the above mentionedchemical substances and compounds. Such a further substance is paraquat.Paraquat is the trivial name for 1,1′-dimethyl-4,4′-bipyridinium, and acommercially available form of paraquat is e.g.1,1′-dimethyl-4,4′-bipyridinium dichloride.

In order to develop new pharmaceutically active compounds, a potentialtarget for medical intervention had to be identified. Thus, processesfor finding pharmaceutically effective compounds include targetidentification. Details for finding a suitable target to deal with HCVinfections are described in WO 02/084294.

Target identification is basically the identification of a particularbiological component, namely a protein and its association withparticular disease states or regulatory systems. A protein identified ina search for a pharmaceutically active chemical compound (drug) that canaffect a disease or its symptoms is called a target. Said target isinvolved in the regulation or control of biological systems and itsfunction can be interfered by with a drug.

The word disease is used herein to refer to an acquired condition orgenetic condition. A disease can alter the normal biological system ofthe body, causing an over or under abundance of chemical compounds(chemical imbalance). The regulatory systems for these chemicalcompounds involve the use by the body of certain proteins to detectimbalances or cause the body to produce neutralizing compounds in anattempt to restore the chemical balance.

The word body is used herein to refer to any biological system, e.g.human, animal, cells, or cell culture.

It is therefore the object of the present invention to providecompounds, compositions and methods which are effective in theprophylaxis and/or treatment of Hepatitis C virus infections, but whichdo not show the negative side-effects described above or at least not tothe extent reported for known products and methods. The object of thepresent invention is solved by the teaching of the independent claims.Further advantageous features, aspects and details of the invention areevident from the dependent claims, the description, and the examples ofthe present application.

DETAILED DESCRIPTION OF THE INVENTION

It has been shown previously that the human cellular protein glutathioneperoxidase-gastrointestinal is specifically downregulated in a body as aresult of HCV infection. This human cellular protein glutathioneperoxidase-gastrointestinal has been identified as a diagnostic andtherapeutic target for dealing with HCV infection.

Glutathione Peroxidase:

Four distinct species of glutathione peroxidase have been identified inmammals to date, the classical cellular enzyme, the phospholipidhydroperoxide metabolizing enzyme, the gastroinestinal tract enzyme andthe extracellular plasma enzyme. Their primary structures are poorlyrelated. It has been shown that they are encoded by different genes andhave different enzymatic properties. The physiological role of the humanplasma enzyme remains still unclear due to the low levels of reducedglutathione in human plasma and the low reactivity of this enzyme.

The human cellular protein glutathione peroxidase-gastrointestinal(GI-GPx) is also known as glutathione peroxidase-related protein 2(GPRP) or glutathione hydrogen peroxide oxidoreductase. It has beenassigned to the Accession Number P18283 and the EC Number 1.11.1.9.

The human cellular protein glutathione peroxidase-gastrointestinal(GI-GPx) catalyzes the reduction of various organic hydroperoxides, aswell as hydrogen peroxide, with glutathione (GSH) as hydrogen donor (2GSH+H₂O₂→GS-GS+2H₂O). It has a molecular weight of 84,000 and consistsof 4 subunits. The enzyme is useful for enzymatic determination of lipidhydroperoxide.

GI-GPx belongs to the family of selenoproteins and plays an importantrole in the defense mechanisms of mammals, birds and fish againstoxidative damage by catalyzing the reduction of a variety ofhydroperoxides, using glutathione as the reducing substrate. It has beensuggested that this enzyme functions in more times as a mechanism ofprotecting the cellular membrane system against peroxidative damage andthat selenium as an essential trace element which may play an importantrole in this suggested function of the enzyme. It is known that bothvitamin E and Se act as antioxidants also in a common mechanism ofoxidative stress as an underlying cause of genetic changes.

Selenium functions within mammalian systems primarily in the form ofselenoproteins. Selenoproteins contain selenium as selenocysteine andperform a variety of physiological roles. Seventeen selenoproteins havebeen identified: cellular or classical glutathione peroxidase; plasma(or extracellular) glutathione peroxidase; phospholipid hydroperoxideglutathione peroxidase; gastrointestinal glutathione peroxidase;selenoprotein P; types 1, 2, and 3 iodothyronine deiodinase;selenoprotein W; thioredoxin reductase; and selenophosphate synthetase.Of these, cellular and plasma glutathione peroxidase are the functionalparameters used for the assessment of selenium status (D. H. Holben, A.M. Smith, J. Am. Diet. Assoc. 1999, 99, 836-843).

Beside vitamin E (DL-α-tocopherol), vitamin C (L-ascorbic acid),co-enzyme Q10, zinc, and selenium a lot of further antioxidants such asN-acetyl-L-cycteine, N-acetyl-S-farnesyl-L-cysteine, Bilirubin, caffeicacid, CAPE, catechin, ceruloplasmin, Coelenterazine, copperdiisopropylsalicylate, deferoxamine mesylate, R-(−)-deprenyl, DMNQ, DTPAdianhydride, Ebselen, ellagic acid, (−)-epigallocatechin,L-ergothioneine, EUK-8, Ferritin, glutathione, glutathionemonoethylester, α-lipoic acid, Luteolin, Manoalide, MCI-186, MnTBAP,MnTMPyP, morin hydrate, NCO-700, NDGA, p-Nitroblue, propyl gallate,Resveratrol, rutin, silymarin, L-stepholidine, taxifolin, tetrandrine,tocopherol acetate, tocotrienol, Trolox®, U-74389G, U83836E, and uricacid (all available from Calbiochem, San Diego, Calif., U.S.A.) whichcan be applied for preventing and/or treating HCV infections bycompensating at least partially the down-regulation of GI-GPx.

Further antioxidants may be selected from the group of carboxylic acidssuch as citric acid and phenolic compounds such as BHA (butylatedhydroxyanisole), BHT (butylated hydroxytoluene), propyl gallate, TBHQ(tert-butyl hydroquinone), tocopherols, lecithin, gums and resin guiac,THBP (trihydroxybutyrophenone), thiodipropionic acid and dilaurylthiodipropionate, and glycines.

Oxidative damage is mainly caused by free radicals, particularlyreactive oxygen intermediates, derived from normal cellular respirationand oxidative burst produced when phagocytic cells destroy bacteria orvirus-infected cells. In order to cope with the constant generation ofpotentially damaging oxygen radicals, eukaryotic organisms have evolvedmany defense mechanisms. These include the above-mentioned antioxidantswhich act as free radicals scavengers and which may interact with GI-GPxand/or may activate, stimulate, and/or increase the expression and/orproduction of GI-GPx. This advantageous effect of the radicals on theamount of GI-GPx generated in the cells competes with the HCV-induceddown-regulation of GI-GPx and supports the cells in their fight againstthe Hepatits C viruses.

HCV Infection Studies:

The only reliable experimental animal HCV infection studies have beenperformed with chimpanzees. So far, there is no simple cell cultureinfection system available or HCV. Although a number of reports havebeen published describing in vitro propagation attempts of HCV inprimary cells and cell lines, questions remain concerningreproducibility, low levels of expression and properly controlleddetection methods (reviewed in J. Gen Virol. 81, 1631; AntiviralChemistry and Chemotherapy 10, 99). Thus, the replicon system describedby Bartenschlager and coworkers (Lohmann et al, Replication ofsubgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285,110. 1999) was used for the studies disclosed herein. This repliconsystem reproduces a crucial part of the HCV replication cycle which isused as a system for simulating HCV infection. Bartenschlager's groupproduced bicistronic recombinant RNAs, so-called “replicons”, whichcarry the neomycin-phosphotransferase (NPT) gene as well as a version ofthe HCV genome where the sequences for the structural HCV proteins weredeleted. After transfection of the subgenomic HCV RNA molecules into thehuman hepatoma cell line HuH-7, cells supporting efficient RNA-dependentRNA replication of the HCV replicons were selected based onco-amplification of the NPT gene and resulting resistance to theantibiotic G-418. Integration of coding information into the cellulargenome was an exclusion criteria for functional replicons. Several lineswere established from G-418 resistant clones with autonomouslyreplicating HCV RNAs detectable by Northern Blotting. Minus-strand RNAreplication intermediates were detected by Northern Blotting ormetabolic radio-labeling, and the production of nonstructural HCVproteins was demonstrated by immuno-precipitation after metaboliclabeling or Western Blotting.

Possible influences and/or dependencies of HCV's RNA-dependent RNAreplication and nonstructural proteins on host cell transcription areaccessible to analysis with the Clontech cDNA arrays used in the methodsdescribed herein. HuH-pcDNA3 cells are HuH7 cells resistant to G-418 byintegration of a NPT gene-carrying plasmid (pcDNA3, Invitrogen) andserve as negative control. Three replicon lines were analyzed forchanges in cellular RNA expression patterns compared to the controlline:

-   -   HuH-9-13: cell line with persistant replicon IRES377/NS3-3′/wt,        described in Science 1999, 285, 110-113,    -   HuH-5-15: cell line with persistant replicon IRES389/NS3-3′/wt,        described in Science 1999, 285, 110-113,    -   HuH-11-7: cell line with persistant replicon IRES377/NS2-3′/wt,        described in Science 1999, 285, 110-113.

These HCV replicon cells serve as a system for simulation of HCVinfected cell systems, especially for simulating HCV infected mammals,including humans. Interference of HCV with the cellular signaling eventsis reflected in differential gene expression when compared to cellularsignaling in control cells. Results from this signal transductionmicroarray analysis revealed significant downregulation of GI-GPx.Radioactively labeled complex cDNA-probes from HCV Replicon cellsHuH-9-13, HuH-5-15, and HuH-11-7 were hybridized to cDNA-arrays andcompared to hybridizations with cDNA-probes from HuH-pcDNA control cellswhich did not contain HCV Replicons.

Based on the surprising results reported herein, one aspect of thepresent invention is directed to specific chemical substances andcompounds useful for the prophylaxis and/or treatment of Hepatitis Cvirus infections. Specifically, these specific chemical substances andcompounds comprise selenium, selenium salts, Vitamine D₃, pegylated andnon-pegylated (standard) α-, β-, and γ-interferon, ribavirin, andretinoids, particularly all forms of retinoic acid, all trans retinoicacid, salts of all trans retinoic acid, C₁-C₁₀ alkyl esters of all transretinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid,C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkylamides of all trans retinoic acid, like 9-cis retinoic acid, salts of9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, saltsof C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkyl amides of9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cis retinoicacid, 13-cis retinoic acid, salts of 13-cis retinoic acid, C₁-C₁₀ alkylesters of 13-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 13-cisretinoic acid, C₁-C₁₀ alkyl amides of 13-cis retinoic acid, salts ofC₁-C₁₀ alkyl amides of 13-cis retinoic acid as well as(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoicacid (TTNPB),(4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)carboxamido]benzoicacid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR) and6-[341-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437;AHPN).

The above mentioned chemical substances and compounds may be combinedwith further compounds like paraquat.

Furthermore, the present invention discloses a method for treatingHepatitis C virus infection in an individual. Preferably the individualis a non-responder to interferon and/or ribavirin therapy. The methodcomprises the step of administering a pharmaceutically effective amountof at least one of the specific chemical compounds and substancesreferred to above, which upregulate at least partially the activity ofGI-GPx or which upregulate at least partially the production of GI-GPxin the cells. To the specific chemical compounds and substances thefurther compounds like all trans retinoic acid and paraquat may beadded.

A similar aspect of the present invention is directed to a method forpreventing and/or treating Hepatitis C virus infection and/or diseasesassociated with HCV infection in cells or cell cultures comprising thestep of administering a pharmaceutically effective amount of at leastone of the specific chemical compounds and substances referred to above,which upregulate at least partially the activity of GI-GPx or whichupregulate at least partially the production of GI-GPx.

Another aspect of the present invention is to provide a method forregulating the production of Hepatitis C virus in an individual or Incells or cell cultures comprising the step of administering apharmaceutically effective amount of at least one of the specificchemical compounds and substances referred to above, which at leastpartially upregulate the activity GI-GPx or which at least partiallyupregulate the production of GI-GPx in the cells. Preferably, theindividual is a non-responder to interferon and/or ribavirin therapy.

In addition to the above-mentioned methods the present invention is alsodirected to a method for preventing and/or treating Hepatitis C virusinfection and/or diseases associated with HCV infection in an individualcomprising the step of administering a pharmaceutically effective amountof at least one of the specific chemical compounds and substancesreferred to above, which activates at least partially GI-GPx or whichactivates or stimulates the production of GI-GPx in the individual.Again, preferably the individual is a non-responder to interferon and/orribavirin therapy.

Another inventive aspect is related to a method for preventing and/ortreating Hepatitis C virus infection and/or diseases associated with HCVinfection in cells or cell cultures comprising the step of administeringa pharmaceutically effective amount of at least one of the specificchemical compounds and substances referred to above, which activate atleast partially the activity of GI-GPx or which activate or stimulate atleast partially the production of GI-GPx.

The term “associated diseases” refers to, for instance, opportunisticinfections, liver cirrhosis, liver cancer, hepatocellular carcinoma, orany other diseases that can come along with HCV infection.

The function of GI-GPx is to detoxify peroxides in cells and protect thecells from oxidative damage. Subjecting HCV infected cells to oxidativestress conditions, preferably induced by paraquat or radicals generatedfrom peroxides, leads to a decreased resistance of HCV infected cells incomparison to uninfected cells against toxicity of radicals. Thus,generating artificial oxidative stress conditions allows selectivekilling of HCV-infected cells.

Examples for useful radical forming compounds (radical initiators) arebipyridyls such as paraquat, 2,2′-bipyridyl and 4,4′-bipyridylderivatives, bis-6-(2,2′-bipyridyl)-pyrimidines,tris-(2,2′-bipyridyl)ruthenium, peroxides such as dibenzoylperoxid,diacetylperoxide, hydrogen peroxide, di-tert.-butylperoxide, or diazacompounds such as diazaisobutyronitril.

Yet another aspect of the present invention is directed to a noveltherapeutic composition useful for the prophylaxis and/or treatment ofan individual afflicted with Hepatitis C virus and/or associateddiseases comprising at least one of the specific chemical substances andcompounds selected from the group consisting of selenium, seleniumsalts, Vitamin D₃, pegylated and non-pegylated (standard) α-, β-, andγ-interferon, ribavirin, and retinoids, particularly all isomeric formsof retinoic acid, like all trans retinoic acid, salts of all transretinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts ofC₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides ofall trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all transretinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, C₁-C₁₀alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of9-cis retinoic acid, C₁-C₁₀ alkyl amides of 9-cis retinoic acid, saltsof C₁-C₁₀ alkyl amides of 9-cis retinoic acid, 13-cis retinoic acid,salts of 13-cis retinoic acid, C₁-C₁₀ alkyl esters of 13-cis retinoicacid, salts of C₁-C₁₀ alkyl esters of 13-cis retinoic acid, C₁-C₁₀ alkylamides of 13-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 13-cisretinoic acid, as well as(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoicacid (TTNPB),(4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)carboxamido]benzoicacid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid(CD437; AHPN). A preferred selenium salt is sodium selenite. Moreover,according to a further aspect of the present invention, the compositionmay contain a certain amount of all trans retinoic acid and paraquat.The preferred individual is a non-responder to interferon and/orribavirin therapy.

Further embodiments of the present Invention are represented by methodsfor regulating the production of Hepatitis C virus in an individual orin cells or cell cultures comprising the step of administering anindividual or the cells a pharmaceutically effective amount of at leastone of the specific chemical substances and compounds selected from thegroup consisting of selenium, selenium salts, Vitamin D₃ and retinoids,like all trans retinoic acid, salts of all trans retinoic acid, C₁-C₁₀alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters ofall trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid,salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoicacid, salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cisretinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid,C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amidesof 9-cis retinoic acid,4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphthalenyl)-1-propenyl]benzoicacid, and/or4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamidobenzoic acid, N-(4-hydroxyphenyl) retinamide (4-HPR), and6-[3-1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437;AHPN), wherein said substance or compound activates or increases atleast partially the activity of said human cellular protein glutathioneperoxidase-gastrointestinal or wherein said agent at least partiallyactivates or stimulates the production of said human cellular proteinglutathione peroxidase-gastrointestinal. The above mentioned chemicalsubstances and compounds may be combined with further compounds like alltrans retinoic acid and paraquat. Preferably, the individual is anon-responder to interferon and/or ribavirin therapy.

Another aspect of the present invention is directed to novel therapeuticcompositions useful within said methods for prophylaxis and/or treatmentof an individual afflicted with Hepatitis C virus and/or associateddiseases. Said compositions comprise at is least one of the specificchemical substances and compounds selected from the group consisting ofselenium, selenium salts, Vitamin D₃ and retinoids, like all transretinoic acid, salts of all trans retinoic acid, C₁-C₁₀ alkyl esters ofall trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all transretinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts ofC₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid,salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoicacid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkylamides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cisretinoic acid,4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphthalenyl)-1-propenyl]benzoicacid, and/or4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamidobenzoic acid, N-(4-hydroxyphenyl)retinamide (4-HPR), and6-[3-1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437;AHPN), capable of increasing the activity of GI-GPx or of activating orstimulating the production and/or expression of GI-GPx. Preferredindividuals are non-responders to interferon and/or ribavirin therapy.

According to still further aspect of the present invention, the noveltherapeutic compositions contain from 0.01 to 0.15% by weight,particularly from 0.02 to 0.05% by weight of the specific chemicalsubstances and compounds or “agent(s)”. The above mentioned chemicalsubstances and compounds may be combined with further compounds like alltrans retinoic acid and paraquat.

Said pharmaceutical compositions may further comprise pharmaceuticallyacceptable carriers, excipients, and/or diluents.

In case the pharmaceutical composition is for oral application,according to a further embodiment of the present invention, thetherapeutic agent (or agents) is (are) administered in the form oftablets or capsules. Such tablets or capsules may contain from 1 to 300mg, preferably from 1 to 150 mg, more preferably from 1 to 100 mg, andparticularly from 1 to 50 mg of the agent or agents.

Another possible way of applying a therapeutically effective amount ofat least one of the above-mentioned specific substances to an individual(patient) is by means of including the substance(s) into liposomes andadministering the liposomes to the individual. Liposomes are sphericalparticles having typically a diameter of about 25 nm to about 5 μm.Liposomes usually comprise one or more concentric lipid double layershaving an aqueous interior compartment (so-called “lipid vesicles”).Liposomes are known as carriers for pharmaceutical substances, which canbe selectively enriched in certain organs and cellular tissues by meansof the liposomes, see e.g. Adv. Drug Deliv. Rev. 19, 425 to 444 (1996)and Science 267, 1275 et seq. (1995).

As used herein, the term “activator” refers to any chemical compoundcapable of upregulating, activating, stimulating, or increasing theamount and/or activity of GI-GPx or its expression.

As used herein, the term “inhibitor” refers to any compound capable ofdownregulating, decreasing, inactivating, suppressing or otherwiseregulating the amount and/or activity of GI-GPx or its expression.Generally, GI-GPx inhibitors may be proteins, oligo- and polypeptides,nucleic acids, such as RNAi, genes, small chemical molecules, or otherchemical moieties.

The term “agent” is used herein as synonym for regulator, inhibitor,and/or activator. Thus, the term “agent” refers to any chemical orbiological compound capable of down- or upregulating, de- or increasing,suppressing or stimulating, inactivating or activating, or otherwiseregulating or effecting the amount and/or activity of GI-GPx and/or theexpression of GI-GPx.

In addition to the role In transmitting genetic information from DNA toproteins, RNA molecules participate actively in many cell processes.Examples are found in translation (rRNA, tRNA, tmRNA), intracellularprotein targeting (SRP), nuclear splicing of pre-mRNA (snRNPs), mRNAediting (gRNA), and X-chromosome inactivation (Xist RNA). Each of theseRNA molecules acts as a functional product in its own right, withoutcoding any protein. Because RNA molecules can fold into unique shapeswith distinct structural features, some RNAs bind to specific proteinsor small molecules (as in the ATP-binding aptamer), while otherscatalyze particular chemical reactions. Thus, RNA aptamers can be usedto interact with GI-GPx and thereby modulate, regulate, activate, orinhibit the activity and biological function of said peroxidase.

As used herein, the term “regulating expression and/or activity”generally refers to any process that functions to control or modulatethe quantity or activity (functionality) of a cellular component. Staticregulation maintains expression and/or activity at some given level.Upregulation refers to a relative increase in expression and/oractivity. Accordingly, downregulation refers to a relative decrease inexpression and/or activity. Downregulation is synonymous with inhibitionof a given cellular component's activity.

Further aspects of the present invention relate to methods either forregulating the expression of the human cellular protein glutathioneperoxidase-gastrointestinal in an Individual or in cells or cellcultures comprising the step of administering either the individual orthe cells or cell cultures a pharmaceutically effective amount of anagent wherein said agent inhibits or decreases at least partially thetranscription of DNA and/or the translation of RNA encoding said humancellular protein glutathione peroxidase-gastrointestinal. Again,preferred individuals are non-responders to interferon and/or ribavirintherapy.

Therapeutics, pharmaceutically active agents or inhibitors,respectively, may be administered to cells from an individual in vitro,or may involve in vivo administration to the individual. The term“individual” preferably refers to mammals and most preferably to humans.Humans particularly preferred are non-responders to interferon and/orribavirin therapy. Routes of administration of pharmaceuticalpreparations to an individual may Include oral and parenteral, includingdermal, intradermal, intragastral, intracutan, intravasal, intravenous,intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal,percutan, rectal, subcutaneous, sublingual, topical or transdermalapplication, but are not limited the these ways of administration. Forinstance, the preferred preparations are in administratable form whichis suitable for oral application. These administratable forms, forexample, include pills, tablets, film tablets, coated tablets, capsules,powders and deposits. Administration to an individual may be in a singledose or in repeated administrations, and may be in any of a variety ofphysiologically acceptable salt forms, and/or with an acceptablepharmaceutical carrier, binder, lubricant, excipient, diluent and/oradjuvant. Pharmaceutically acceptable salt forms and standardpharmaceutical formulation techniques are well known to persons skilledin the art.

As used herein, a “pharmaceutical effective amount” of a GI-GPxactivator is an amount effective to achieve the desired physiologicalresult, either in cells or cell cultures treated in vitro or in asubject (e.g. individual, particularly human being) treated in vivo.Specifically, a pharmaceutically effective amount is an amountsufficient to inhibit, for some period of time, one or more of theclinically defined pathological processes associated with the viralinfection. The effective amount may vary depending on the specificGI-GPx inhibitor or activator selected, and is also dependent on avariety of factors and conditions related to the subject to be treatedand the severity of the infection. For example, if the activator is tobe administered in vivo, factors such as the age, weight and health ofthe patient as well as dose response curves and toxicity data obtainedin pre-clinical animal work would be among those considered. If theactivator is to be contacted with the cells or cell cultures in vitro,one would also design a variety of pre-clinical in vitro studies toassess such parameters as uptake, half-life, dose, toxicity, etc. Thedetermination of a pharmaceutically effective amount for a given agentis well within the ability of those skilled In the art. As mentionedabove, a “therapeutically effective amount” of the substances andcompounds according to the present invention may be 0.01 to 0.15% byweight of a pharmaceutical composition. In case a tablet or capsule isused as administrative form, the amount of the effective substance orcompound in the tablet or capsule may be 1 to 300 mg, preferably 1 to150 mg, more preferably 1 to 100 mg, and particularly 1 to 50 mg.

The present disclosure teaches for the first time the upregulation ofGI-GPx specifically involved in the viral infection of Hepatitis C virususing specific chemical compounds and substances selected from the groupconsisting of selenium, selenium salts, Vitamin D₃, pegylated andnon-pegylated (standard) α-, β-, and γ-interferon, ribavirin, andretinoids, particularly all isomeric forms of retinoic acid, like alltrans retinoic acid, salts of all trans retinoic acid, C₁-C₁₀ alkylesters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of alltrans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid,salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoicacid, salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cisretinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid,C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amidesof 9-cis retinoic acid, 13-cis retinoic acid, salts of 13-cis retinoicacid, C₁-C₁₀ alkyl esters of 13-cis retinoic acid, salts of C₁-C₁₀ alkylesters of 13-cis retinoic acid, C₁-C₁₀ alkyl amides of 13-cis retinoicacid, salts of C₁-C₁₀ alkyl amides of 13-cis retinoic acid, as well as4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphthalenyl)-1-propenyl]benzoicacid, and/or4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamidobenzoic acid, N-(4-hydroxyphenyl)retinamide (4-HPR), and6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid(CD437; AHPN). The above mentioned chemical substances and compounds maybe combined with further compounds like all trans retinoic acid andparaquat.

The polypeptide product of gene expression may be assayed to determinethe amount of expression as well. Methods for assaying for a proteininclude, but are not limited to, Western Blotting, immuno-precipitation,radioimmuno assay, immunohistochemistry and peptide immobilization in anordered array. It is understood, however, that any method forspecifically and quantitatively measuring a specific protein or mRNAproduct can be used.

The present invention further incorporates by reference in theirentirety techniques well known in the field of microarray constructionand analysis. These techniques include, but are not limited to,techniques described in the following patents and patent applicationsdescribing array of biopolymeric compounds and methods for theirfabrication:

-   -   U.S. Pat. Nos. 5,242,974; 5,384,261; 5,405,783; 5,412,087;        5,424,186; 5,429,807; 5,436,327; 5,445,934; 5,472,672;        5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,556,752;        5,561,071; 5,559,895; 5,624,711; 5,639,603; 5,658,734;        5,807,522; 6,087,102; WO 93/17126; WO 95/11995; WO 95/35505; EP        742 287; and EP 799 897.

Techniques also include, but are not limited to, techniques described inthe following patents and patent application describing methods of usingarrays in various applications:

-   -   U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049;        5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464;        5,547,839; 5,580,732; 5,661,028; 5,994,076; 6,033,860;        6,040,138; 6,040,140; WO 95/21265; WO 96/31622; WO 97/10365; WO        97/27317; EP 373 203; and EP 785 280

A robust cell culture system for the hepatitis C virus (HCV) has notbeen established. For this reason, it is extremely difficult to studyhow HCV infects cells and to test anti-viral drugs in a model system(the only animals that can be infected are humans and chimpanzees). Amajor step in devising a culture system for HCV was established by thereplicon cell lines (Lohmann, V., Korner, F., Koch, J.-O., Herian, U.,Theilmann, L., and Bartenschlager, R. 1999. Replication of subgenomichepatitis C virus RNAs in a hepatoma cell line. Science. 285: 110-113).Replication of subgenomic HCV RNAs in cultured hepatocytes were obtainedfor the first time. These subgenomic replicons are composed of only thepart of the HCV genome that encodes the non-structural proteins but arecompetent to be replicated in cells and synthesize viral proteins. Thereplicons described in the scientific article of Lohmann et al. citedabove and used for the present investigation allows studies of HCVreplication, pathogenesis and evolution in cell culture. They may alsoallow for cell-based testing of certain types of anti-viral drugs.

Recently, gastrointestinal-glutathione peroxidase (GI-GPx) could bevalidated as target in HCV-replication (see WO 02/084294). As mentionedabove, GI-GPx belongs to the family of selenoproteins and plays animportant role in the defense mechanisms of eucaryotic cells againstoxidative damage by catalyzing the reduction of a variety ofhydroperoxides, using glutathione as the reducing substrate. It has beensuggested that this enzyme functions as a mechanism of protecting thecellular membrane system against peroxidative damage. Selenium as anecessary trace element suggests the essential function of this enzyme.

Selenium functions within mammalian systems primarily in the form ofselenoproteins. Selenoproteins contain selenium as selenocysteine andperform a variety of physiological roles. Seventeen selenoproteins havebeen identified: cellular or classical glutathione peroxidase; plasma(or extracellular) glutathione peroxidase; phospholipid hydroperoxideglutathione peroxidase; gastrointestinal glutathione peroxidase;selenoprotein P; types 1, 2, and 3 iodothyronine deiodinase;selenoprotein W; thioredoxin reductase; and selenophosphate synthetase.Of these, cellular and plasma glutathione peroxidase are the functionalparameters used for the assessment of selenium status (D. H. Holben, A.M. Smith, J. Am. Diet. Assoc. 1999, 99, 836-843).

GI-GPx is drastically down-regulated in HCV replicon cells compared withmock-transfected HuH7 cells. Forcing replicon cells to re-express GI-GPx(e.g. by infection with GI-GPx containing Adenovirus) results inreduction of subgenomic HCV RNA and of the HCV protein NS5a to hardlydetectable levels (see WO 02/084294). According to the present inventionthe knowledge of this inverse correlation was used to develop a methodto up-regulate the expression of the cellular, endogenous GI-GPx gene.This up-regulation in replicon cells causes a depletion of HCV.

It is readily apparent to those skilled in the art that other suitablemodifications and adaptations of the compositions and methods of theinvention described herein are evident and may be made without departingfrom the scope of the invention or the embodiments disclosed herein.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included for purposes of illustration only and are not intended tolimit the invention.

EXAMPLES

Reference is Made to the Examples of WO 02/084294, which areIncorporated herein by Reference.

Moreover, as model system for HCV replication there were utilized threereplicon cell lines provided by Prof. R. Bartenschlager (University ofHeidelberg, FRG). Cultures were treated for various periods of time withall bans retinoic acid (RA) for comparative purposes and the otheragents selenium, selenium salts, Vitamin D₃ and retinoids, like 9-cisretinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkylamides of 9-cis retinoic add, N-(4-hydroxyphenyl)retinamide (4-HPR) and6-[3-(1-adamantyl)+hydroxyphenyl]-2-naphthalene carboxylic acid (CD437;AHPN) (obtained from Sigma). Levels of expression of GI-GPx was measuredon protein level by Western Blotting using antibodies provided by Prof.Brigelius-Flohe (University of Potsdam, FRG) and on RNA level byNorthern blotting using GI-GPx-specific oligonucleotides as probes.Levels of HCV RNA were investigated by Northern Blotting using a DNAoligonucleotide complementary to the neomycin phosphotansferase gene asprobe. Concentration of the viral protein NS5a was determined by WesternBlotting with an NS5a-specific antibody (Biogenesis, UK).

Treatment of replicon cells for three days with all trans retinoic acid(1 μM) had hardly an effect on GI-GPx and HCV expression. However, afterseven days of incubation, a drastic up-regulation of GI-GPx on RNA- andprotein level (three- to ten-fold) was observed. Concomitantlyexpression of subgenomic HCV RNA and of viral protein NS5a wasdownregulated two- to five-fold, depending on the cell lineinvestigated. Furthermore, surprisingly it was found that a furtherdownregulation of HCV-RNA and -NS5a protein was dependent on theaddition of selenium or a selenium salt, e.g. sodium selenite (50 nM).This fact implies, that downregulation of HCV was promoted firstly byactivation of the GI-GPx gene on transcriptional level by retinoic acidand secondly by the synthesis of selenoprotein(s) for which sodiumselenite was needed. Indeed it could be shown that all trans retinoicacid-induced downregulation of HCV is independent of the innate immuneresponse induced by interferon. Thus, all trans retinoic acid did notinduce the transcription of PKR (double strand RNA-dependent proteinkinase). Severe cytotoxic effects were neither observed for all transretinoic acid nor for sodium selenite, or both in combination.

The presented findings show that retinoids (in combination with seleniumor selenium salts like sodium selenite and/or CAMP and/or analoguesthereof) can be used for the treatment of HCV-positive patients.Especially the usage of retinoids with high specificity for induction ofthe GI-GPx, like N-(4-hydroxyphenyl) retinamide (4-HPR) and6-[3-1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437;AHPN), are preferred. 4-HPR and AHPN display significant potential astherapeutic agents in the prophylaxis and treatment of a number ofpremalignant and malignant conditions in the context of HCV infections.Indeed, the obtained data show that next to all trans retinoic acidother nuclear receptor ligands, like 9-cis retinoic acid as well assalts thereof, 9-cis retinoic acid C₁ to C₁₀ alkyl esters as well assalts therof, 9-cis retinoic acid C₁ to C₁₀ alkyl amides as well assalts therof, and Vitamin D₃, are also capable of reducing HCV load.

All-trans retinoic acid on replicon cells for six days led to anupregulation of GI-GPx RNA and protein due to the fact that theGI-GPx-promoter contains three retinoic acid receptor recognitionelements. In the presence of selenium or a selenium salt like sodiumselenite a two- to five-fold reduction of HCV-RNA and HCV-NS5a proteinwas observed in the absence of toxic effects. Moreover, also thespecific retinoids, like N-(4-hydroxyphenyl)retinamide (4-HPR) and6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid(CD437; AHPN), 9-cis retinoic acid, 9-cis retinoic acid C₁ to C₁₀ alkylesters, 9-cis retinoic acid C₁ to C₁₀ alkyl amides, and Vitamin D₃ aloneor in combination with each other or with selenium or a selenium saltshowed a similar effect.

Moreover, first preliminary result have shown that 9-cis retinoic acidand its above-mentioned alkyl and amide derivates downregulate HCV RNAsignificantly better than all trans retinoic acid alone.

The following examples describing administrative forms for a lotion(solution), gel, cream, soft gelatin capsules, hard gelatine capsules,tablets, and sachets containing 9-cis retinoic acid are taken from U.S.Pat. No. 5,428,071 and EP-B1-0 552 624.

Example 1

Lotion (solution) preferred 9-cis-Retinoic Acid 0.02-0.30 g PropyleneGlycol 5.00-20.00 g 10.00 g PEG-Glyceryl Cocoate* 0.00-20.00 g 10.00 gdl-alpha-Tocopherol 0.001-0.50 g 0.02 g Ascorbyl Palmitate 0.01-0.20 g0.10 g Propyl Gallate 0.001-0.02 g 0.002 g Citric acid, anhydr**0.00-0.20 g 0.01 g Isopropanol*** 40.00-90.00 g 50.00 g Water, dem. ad100.00 g 100.00 g (resp. ml)*or other tensides**or other complexing agents, e.g. EDTA***or other alcohols, e.g. Ethanol

Example 2

Gel preferred 9-cis-Retinoic Acid 0.02-0.30 g Propylene Glycol5.00-20.00 g 10.00 g PEG-Glyceryl Cocoate* 0.00-20.00 g 10.00 gdl-alpha-Tocopherol 0.001-0.50 g 0.02 g Ascorbyl Palmitate 0.01-0.20 g0.10 g Propyl Gallate 0.001-0.02 g 0.002 g Citric acid, anhydr**0.00-0.20 g 0.01 g Isopropanol*** 40.00-90.00 g 50.00 g HPMC****0.50-5.00 g 3.0 g Preservative***** q.s. q.s. Water, dem. ad 100.00 g100.00 g*or other tensides**or other complexing agents, e.g. EDTA***or other alcohols, e.g. Ethanol****Hydroxypropyl Methylcellulose or other polymers e.g. neutralizedCarbomer, Methyl Cellulose, Sodium Carboxymethylcellulose*****Preservatives, e.g. Paraben esters (methyl, ethyl, propyl, butyl),Sorbic Acid, Benzoic, Acid

Example 3

Cream preferred 9-cis-Retinoic Acid 0.02-0.30 g Glycerol 0.00-10.00 g5.00 g Na.sub.2 EDTA 0.001-0.50 g 0.03 g Glycerides* 5.00-20.00 g 10.00g Cetyl Alcohol 0.50-5.00 g 1.00 g Stearyl Alcohol 0.50-5.00 g 1.00 gGlycerol mono Stearate 1.00-8.00 g 4.00 g Cetaereth** 0.50-5.00 g 2.00 gdl-alpha-Tocopherol 0.001-0.50 g 0.02 g Preservative*** q.s. q.s. Water,dem. ad 100.00 g 100.00 g*e.g. Caprylic/Capric/Triglyceride, Caprylic/Capric/LinoleicTriglyceride natural glycerides, as well as e.g., Propylene Glycol,Dicaprylate/Dicaprate and waxes such as Stearyl Stearate, Oleyl Oleate,Isopropyl Myristate.**Ceteareth 5-30, or other emulsifiers such as Polysorbate 20-80,Sorbitane esters of fatty acids, fatty acid esters of PEG.***Preservatives e.g., Paraben esters (methyl, ethyl, propyl, butyl),Sorbic Acid, Benzoic Acid.

Example 4

Fill mass for soft gelatin capsules 9-cis-Retinoic Acid 5.00-50.00 mgOil* 1-3 parts Wax mixture** 1-5 parts Fill volume 1-6 minims*natural vegetable oils, e.g., soy oil, peanut oil, and artificialglycerides**composition of natural and artificial waxes or partially hydrated fa

Example 5

1. Hard Gelatine capsules containing 20 mg active substance:Composition: One Capsule contains: 9-cis-Retinoic acid 20.0 mg. GelatineBloom 30 70.0 mg. Maltodextrin MD 05 108.0 mg. dl-alpha-Tocopherol 2.0mg. Sodium ascorbate 10.0 mg. Microcrystalline cellulose 48.0 mg.Magnesium stearate 2.0 mg. (weight capsule content) 260.0 mg. Procedure:The active substance is wet milled in a solution of gelatine,maltodextrin, dl-alpha-Tocopherol and sodium ascorbate. The wet milledsuspension is spray-dried. The spray-dried powder is mixed withmicrocrystalline cellulose and magnesium stearate. 260 mg. each of thismixture are filled into hard gelatine capsules of suitable size andcolor.

Example 6

2. Tablet containing 20 mg active substance: Composition: Tablet kernel:9-cis-Retinoic acid 20.0 mg Anhydrous lactose 130.5 mg MicrocrystallineCellulose 80.0 mg dl-alpha-Tocopherol 2.0 mg Sodium ascorbate 10.0 mgPolyvinylpyrrolidone K30 5.0 mg Magnesium stearate 2.5 mg (Kernelweight) 250.0 mg Film coat: Hydroxypropyl methylcellulose 3.5 mgPolyethylenglycol 6000 0.8 mg Talc 1.3 mg Iron oxide, yellow 0.8 mgTitanium dioxide 0.8 mg (weight of the film) 7.4 mg Procedure:9-cis-Retinoic acid is mixed with anhydrous lactose and micro-crystalline cellulose. The mixture is granulated in water with asolution/dispersion of polyvinylpyrrolidone, dl-.alpha.-Tocopherol andsodium ascorbate. The granular material is mixed with magnesium stearateand afterwards pressed as kernels with 250 mg. weight. The kernels arefilm coated with a solution/suspension of above-mentioned composition.

Example 7

Sachet Containing 50 mg Active Substance Composition: 9-cis-Retinoicacid 50.0 mg Lactose, fine powder 990.0 mg Microcrystalline Cellulose1400.0 mg Sodium Carboxymethyl-cellulose 14.0 mg dl-alpha-Tocopherol 5.0mg Sodium ascorbate 20.0 mg Polyvinylpyrrolidone K30 10.0 mg Magnesiumstearate 10.0 mg Flavouring Agents 1.0 mg (Fill weight of a sachet)2500.0 mg Procedure: 9-cis-Retinoic acid is mixed with lactose,microcrystalline cellulose and sodium carboxymethyl cellulose. Themixture is granulated in water with a solution/dispersion ofpolyvinylpyrrolidone, dl-alpha-Tocopherol and sodium ascorbate. Thegranule is mixed with magnesium stearate and flavoring agents. It isfilled into sachets of suitable size.

In the following, results of tests are presented relating to retinoicacid and derivatives thereof for treatment of HCV infected cellsnon-responding to interferon treatment.

Treating the HCV replicon cell lines with all trans retinoic acid(ATRA), or some derivatives thereof, resulted in suppression of HCV RNAand of NS5a protein expression (FIG. 1, Panel A). Many findingsdemonstrate that the underlying mechanism is due to the up-regulation ofthe selenocystein protein gastrointestinal glutathione peroxidase(GI-GPx) gene (FIG. 1, Panel B). E.g., this effect was most prominentwhen sodium selenite (50 nM) was given to the cell culture medium (FIG.1, Panel A), which is needed for the synthesis and thus up-regulation ofthe GI-GPx protein (FIG. 1, Panel B).

Retinoic acid (RA)- and Interferon (IFN)-Dependent Pathways

In the course of retinoic acid studies it was investigated whether theRA-effect may also be mediated by activating an interferon response. Atypical interferon-inducible gene codes for the double-strandedRNA-dependent protein kinase (PKR). Mock-transfected HuH7 cells (pcDNA3)and replicon cell lines were treated with ATRA (1 μM), but noup-regulation of the PKR mRNA levels monitored by Northern blotting wasobserved. IFN as a control, however, caused a dramatic up-regulation ofPKR mRNA. An example for a replicon cell line is shown in FIG. 1, PanelC.

These results show that RA acts independently of IFN on the repliconsystem. These results furthermore imply that HCV-patients, who do notreact on IFN treatment, so called non-responders, may be cured byRA-treatment.

Unfortunately, there is no IFN-resistant replicon system described inthe literature to test this hypothesis directly.

Effect of RA in Combination with IFN

In a recent publication (Retinoic acid enhances the antiviral effect ofinterferon on hepatitis C virus replication through increased expressionof type I interferon receptor. Hamamoto et al., J. Lab. Clin. Med. 141,2003, 58-66) it is asserted that treating HuH7 cells with ATRA and 9-cisRA induces the expression of Interferon Type I receptor subunits (max.2-fold after 24 hrs on RNA level, TaqMan analysis). Interestingly, thiseffect was independent of the dose (FIG. 2 of the article by Hamamotocited above). IFN treatment decreased the concentration of transfectedHCV (replicon-) RNA, and this effect was enhanced by treatment with RAs.The authors conclude that RAs increase the anti-HCV replication effectof IFN-alpha through up regulation of type I IFN-receptor in HuH-7cells.

The expression of interferon receptors after treatment of replicon cellswith ATRA was analyzed by western Blotting, but no up-regulation ofinterferon receptors on protein level was observed.

When using sub-therapeutic concentrations of IFN alpha, we found adose-dependent reduction of the HCV protein NS5a (FIG. 2, left Panel).In the presence of RA, the HCV down-regulation was slightly enhanced(center Panel). The strongest effect was observed, when IFN was appliedwith ATRA and sodium selenite together (FIG. 2, right Panel).

The data show that the IFN and RA-effects are additive and imply thattheir mechanisms to down-regulate HCV are independent. This findingfurther substantiates the hypothesis that RA can act in IFN-resistantpatients (non-responders).

Comparing the results presented by Hamamoto with the results describedherein reveals the following:

Both groups come to the same conclusion, namely that IFN and RA promoteadditive anti-HCV effects. However, while Hamamoto asserts that the RAeffect is due to the up-regulation of IFN Type I receptor (a findingwhich could not be reproduced), according to the present invention it isbelieved that the RA-effect is due to the up-regulation of GI-GPx.

Furthermore, the ability of several retinoids (i.e. all trans retinoicacid (ATRA); 9-cis retinoic acid (9-cis RA); 13-cis retinoic acid(13-cis RA);(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoicacid (TTNPB);(4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)carbox-amido]benzoicacid (AM-580); and N-(4-hydroxyphenyl)retinamide (4-HPR)) to act asligands for nuclear receptors (RAR=Retinoic Acid Receptor; RXR=RetinoidX Receptor) was tested and compared with a non retinoid substance(11-methoxy-3,7,11-trimethyl-2E,4E-dodecadienoic acid=methoprene acid).The results are shown in the following Table 1. TABLE 1 LigandReceptor(s) Effect on Replication ATRA RAR (Kd* = 0.2-0.4 nM) +++ 9-cisRA RAR, RXR +++ 13-cis RA RAR, RXR +++ TTNPB RAR (EC₅₀** = 2-21 nM) +++4-HPR RAR ++ AM-580 RARα/not RXR ++ Methoprene acid RXR −*Kd = dissociation constant**EC₅₀ = effector concentration (concentration showing 50% effect)+++ = very strong inhibitory effect;++ = medium inhibitory effect;− = no inhibitory effect

The data imply that RARs, but not RXRs are involved in up-regulation ofthe glutathione peroxidase-gastrointestinal (GI-GPx) promoter. The datafurthermore show that inhibition of HCV replication was linked toup-regulation of GI-GPx mRNA.

Preliminary studies have shown that retinoic acids (e.g. Vesanoid®,Roche Pharmaceuticals, Nutley, N.J., USA) or retinoids may beadministered to a patient in amount of about 1 to 100 mg/m²/day,preferably 20 to 80 mg/m²/day, more preferably 30 to 60 mg/m²/day, andparticularly 40 to 50 mg/m²/day. Suitable doses are 1 to 4 timer perday, preferably 1 to 3 times per day, and particularly 2 times a day.

If a combination therapy is applied, in addition to the retinoic acid orretinoid in the amounts mentioned above, an interferon (e.g. pegylatedinterferon α, e.g. Pegasys® (Hoffmann-La Roche)) may be administered inan amount of about 135 to 180 μg/week (preferably 1 dose per week).

Specifically preferred is a therapy with ATRA alone for the treatment ofHCV infections of non responders. Moreover, the treatment with ATRA plusinterferon (pegylated or non-pegylated α, β, or γ-interferon), ATRA plusinterferon (pegylated or non-pegylated α, β, or γ-interferon) plusselenium or selenium salt, or ATRA plus selenium (and/or selenium salt)and ribavirin is particularly preferred.

1. A composition useful for the prophylaxis and/or treatment of an individual afflicted with a Hepatitis C virus (HCV) infection and/or at least one disease associated with a HCV infection, said composition comprising at least one agent selected from selenium, selenium salts, Vitamin D₃, all trans retinoic acid, salts of all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl) retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN).
 2. The composition according to claim 1, wherein the composition comprises from 0.01 to 0.15% by weight of the agent(s).
 3. The composition according to claim 1, wherein the composition comprises from 0.02 to 0.05% by weight of the agent(s).
 4. The composition according to claim 1, wherein the selenium salt is sodium selenite.
 5. The composition according to claim 1, wherein the composition further comprises at least one of the following compounds: pegylated α-, β-, and/or γ-interferon, non-pegylated (standard) α-, β-, and/or γ-interferon, and ribavirin.
 6. The composition according to claim 1, wherein the composition further comprises paraquat.
 7. The composition according to claim 1, further comprising at least one pharmaceutically acceptable carrier, excipient and/or diluent.
 8. The composition according to claim 1, wherein the individual afflicted with a HCV infection and/or at least one disease associated with HCV infection is a non-responder to interferon and/or ribavirin therapy.
 9. A method for regulating the production of Hepatitis C virus in an individual and/or for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection in an individual, the method comprising administering a pharmaceutical composition comprising a pharmaceutically effective amount of at least one agent selected from selenium, selenium salts. Vitamin D₃, all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ allyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) to the individual.
 10. The method according to claim 9, wherein the pharmaceutical composition comprises from 0.01 to 0.15% by weight of the agent(s).
 11. The method according to claim 10, wherein the pharmaceutical composition comprises from 0.02 to 0.05% by weight of the agent(s).
 12. The method according to claim 9, wherein the selenium salt is sodium selenite.
 13. The method according to claim 9, wherein the pharmaceutical composition further comprises at least one of the following compounds: pegylated α-, β, and/or γ-interferon, non-pegylated (standard) α, β-, and/or γ-interferon, and ribavirin.
 14. The method according to claim 9, wherein the pharmaceutical composition further comprises paraquat.
 15. The method according to claim 9, wherein said pharmaceutical composition is for oral application.
 16. The method according to claim 9, wherein said pharmaceutical composition is for topical application.
 17. The method according claim 15, wherein an oral dosage unit of said pharmaceutical composition contains from 1 to 300 mg, preferably 1 to 150 mg, more preferably from 1 to 100 mg, and particularly from 1 to 50 mg of the agent(s).
 18. The method according to claim 9, wherein the individual is a non-responder to interferon and/or ribavirin therapy.
 19. The composition of claim 1, wherein said composition is in unit dosage form for oral administration, further comprising a pharmaceutically acceptable carrier suitable for oral administration, said agent(s) being present in said unit dosage form in an amount of from about 1 to 50 mg wherein said unit dosage form is a tablet or capsule.
 20. The composition of claim 19, further comprising paraquat. 21-22. (canceled)
 23. A method for regulating the production of Hepatitis C virus in cells or cell cultures and/or for preventing and/or treating Hepatitis C virus infection and/or diseases associated with HCV infection in cells or cell cultures, the method comprising administering a pharmaceutically effective amount of an agent selected from selenium, selenium salts, Vitamin D₃, all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, salts of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) to the cells or cell culture. 24-26. (canceled)
 27. The method according to claim 23 or 55, further comprising administering paraquat.
 28. The method of claim 9, wherein said composition is in a unit dosage form the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
 29. The method according to claim 28, wherein the composition further comprises at least one of the compounds all trans retinoic acid, pegylated α-, β-, and/or γ-interferon, non-pegylated (standard) α, β-, and/or γ-interferon, and ribavirin.
 30. The method according to claim 28, wherein the selenium salt is sodium selenite.
 31. The method according to claim 28, wherein the composition further comprises paraquat.
 32. The method according to claim 28, wherein said composition is for oral application.
 33. The method according to claim 32, wherein the unit dosage form for oral application is a tablet or capsule.
 34. The method according to claim 33, wherein the tablet or capsule comprises between 1 and 300 mg, preferably between 1 to 150 mg, more preferably between 1 to 100 mg, and particularly between 1 and 50 mg of the agent.
 35. The method according to claim 28, wherein said composition is for topical application. 36-41. (canceled)
 42. A method for regulating the expression of the human cellular protein glutathione peroxidase-gastrointestinal in an individual comprising administering to the individual a pharmaceutically effective amount of an agent selected from selenium, selenium salts, Vitamin D₃, all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN), wherein said agent inhibits or activates at least partially the transcription of DNA and/or the translation of RNA encoding said human cellular protein glutathione peroxidase-gastrointestinal.
 43. The method according to claim 42, wherein the individual is a non-responder to interferon and/or ribavirin therapy. 44-45. (canceled)
 46. A method for regulating the expression of the human cellular protein glutathione peroxidase-gastrointestinal in cells or cell culture comprising administering to the cells or cell culture a pharmaceutically effective amount of an agent selected from selenium, selenium salts, Vitamin D₃, all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, C₁-C₁₀ alkyl amide of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amide of 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN), wherein said agent activates at least partially the transcription of DNA and/or the translation of RNA encoding said human cellular protein glutathione peroxidase-gastrointestinal.
 47. A method for regulating the activity of the human cellular protein glutathione peroxidase-gastrointestinal in an individual comprising administering to the individual a pharmaceutically effective amount of an agent selected from selenium, selenium salts, Vitamin D₃, all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, C₁-C₁₀ alkyl amide of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amide of 9-cis retinoic acid, C₁-C₁₀ alkyl amide of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amide of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN), wherein said agent interacts with said human cellular protein glutathione peroxidase-gastrointestinal.
 48. The method according to claim 47, wherein the individual is a non-responder to interferon and/or ribavirin therapy.
 49. A method for regulating the activity of the human cellular protein glutathione peroxidase-gastrointestinal in cells or cell culture comprising administering to the cells or cell culture a pharmaceutically effective amount of an agent selected from selenium, selenium salts, Vitamin D₁₃, all trans retinoic acid, C₁-C₁₀ alkyl esters of all trans retinoic acid, salts of C₁-C₁₀ alkyl esters of all trans retinoic acid, C₁-C₁₀ alkyl amides of all trans retinoic acid, salts of C₁-C₁₀ alkyl amides of all trans retinoic acid, 9-cis retinoic acid, C₁-C₁₀ alkyl esters of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl esters of 9-cis retinoic acid, C₁-C₁₀ alkyl amides of 9-cis retinoic acid, salts of C₁-C₁₀ alkyl amides of 9-cis retinoic acid, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1-propenyl]benzoic acid (TTNPB), (4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (AM-580), N-(4-hydroxyphenyl)retinamide (4-HPR), and 6-[3-(1-adamantyl)₄-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN), wherein said agent interacts with said human cellular protein glutathione peroxidase-gastrointestinal.
 50. The method according to any one of claims 42, 46, 47, and 49, further comprising administering paraquat.
 51. The composition according to claim 5, wherein the individual afflicted with a HCV infection and/or at least one disease associated with HCV infection is a non-responder to interferon and/or ribavirin therapy.
 52. The method according to claim 15, wherein the individual is a non-responder to interferon and/or ribavirin therapy.
 53. The method according to claim 9, wherein said agent activates at least partially the activity of the human cellular protein glutathione peroxidase-gastrointestinal or which activates or stimulates at least partially the production of said human cellular protein glutathione peroxidate-gastrointestinal.
 54. The method according to claim 13, wherein said agent activates at least partially the activity of the human cellular protein glutathione peroxidase-gastrointestinal or which activates or stimulates at least partially the production of said human cellular protein glutathione peroxidase-gastrointestinal.
 55. The method according to claim 18, wherein said agent activates at least partially the activity of the human cellular protein glutathione peroxidase-gastrointestinal or which activates or stimulates at least partially the production of said human cellular protein glutathione peroxidase-gastrointestinal.
 56. The method according to claim 23, wherein said agent activates at least partially the activity of the human cellular protein glutathione peroxidase-gastrointestinal. 